Detection of methicillin-resistant and methicillin-sensitive staphylococcus aureus in biological samples

ABSTRACT

Disclosed are methods of identifying a methicillin-resistant  Staphylococcus aureus  (MRSA) or methicillin-sensitive  Staphylococcus aureus  (MSSA) in a sample. The present invention provides a diagnostic method comprising modification of sequences of  S. aureus  by converting non-methylated cytosine residues ultimately into thymidine residues in the target nucleic acid. The invention further provides for the detection of modified sequences derived from the spa gene, the mecA gene, and the integrated SCCmec cassette of  S. aureus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/962,846, filed Jul. 31, 2007, hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to methods of pathogendetection. In particular, the present invention relates to methods ofdetecting methicillin-sensitive (MSSA) and/or methicillin-resistantStaphylococcus aureus (MRSA) in a biological sample.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Staphylococcus aureus (S. aureus) is a cause of a variety of conditionsin humans, including skin infections (e.g. folliculitis, styes,cellulitis, impetigo, and furuneulosis), pneumonia, mastitis, phlebitis,meningitis, scalded skin syndrome, osteomyelitis, urinary tractinfections, and food poisoning. S. aureus is also a major cause ofhospital-acquired (HA or nosocomial) infection of surgical wounds.Therefore, it is desirable to have a diagnostic assay to detect S.aureus. Additionally, methicillin-resistant S. aureus (MRSA) has emergedand become exceedingly prevalent as a nosocomial pathogen. Therefore, itis also desirable to have a diagnostic assay that distinguishesmethicillin-sensitive strains of S. aureus (MSSA) frommethicillin-resistant strains.

MRSA is one of the two “most out of control” antibiotic resistantpathogens; vancomycin-resistant enterococcus is the other (Society forHealthcare and Epidemiology, SHEA guidelines 2003). Over 50% ofnosocomial infections in intensive care units are due to MRSA (NationalNosocomial Infections Surveillance System, NNIS report, January1992-June 2004). Accordingly, MRSA represents a significant threat topublic health.

Hospital acquired (HA) MRSA is typically controlled by monitoringpatients and personnel for infection. Contact precautions and/or patientisolation may be appropriate when an infection develops or to preventinfections to individuals particularly at risk. The prevalence ofCommunity acquired (CA) MRSA is also increasing. CA-MRSA is defined asMRSA acquired in persons with no known risk factors for MRSA infection(e.g. recent hospitalization, contact with infected patient). Inclinical activities, the quick and reliable identification of MRSA hasbecome important for the diagnosis and treatment of infected patients aswell as for implementation and management of hospital infection controlprocedures.

Methicillin resistance is caused by the acquisition of an exogenous genemecA that encodes penicillin-binding protein (PBP2a or PBP2′). mecA iscarried on a mobile genetic element called Staphylococcal cassettechromosome mec (SCCmec) which also contains the ccr gene complexencoding the recombinases necessary for the element's mobility. TheSCCmec cassette is a large element that can move in and out of the S.aureus genome. SCCmec integrates at a specific site (attBscc) near thechromosomal origin of replication of S. aureus within the 3′ end of theorfx gene, which has no known function. There are a variety of differenttypes of SCCmec defined by variability in length (approximately 20-60kb), gene content and other factors such as ccr gene complex type.

The mecA gene is also present in coagulase-negative Staphylococcus (CNS)strains that are less pathogenic than S. aureus. These strains includeS. epidermidis, S. haemolyticus, S. sapropliyticus, S. capitis, S.warneri, S. sciuri and S. caprae. Some of these other strains ofStaphylococcus inhabit the same environments as S. aureus such as theanterior nares and the skin. It follows that clinical samples such asnasal swabs or wound swabs could potentially contain a mixture of morethan one Staphylococcal species, Therefore, detection of mecA alone isnot sufficient to identify MRSA directly from clinical sample. Becauseidentification of MRSA is of greater clinical significance than theother Staphylococcus species due to its increased pathogenicity andtoxicity, it is desirable that a diagnostic assay distinguish MRSA fromthe other staphylococcal strains containing the mecA gene.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide methodsfor detecting MRSA and/or MSSA in a biological sample. In particular,the present methods relate to the positive identification of MRSA and/orMSSA using detection of three gene markers. The present methods alsorelate to converting the nucleic acids in a sample so that unmethylatedcytosine residues are replaced by thymidine residues and then todetecting sequence-modified MRSA and/or MSSA gene markers in theconverted sample.

In some aspects, the present invention provides methods of detectingsequence-modified nucleic acids from Staphylococcus aureus in abiological sample, comprising converting the unmethylated cytosinespresent in the nucleic acids contained in the biological sample touracils to produce sequence modified nucleic acids, and then bringingthe biological sample containing the sequence modified nucleic acids incontact with one or more primer pairs that may be used to distinguishMRSA from MSSA and other Staphylococcal strains. In one embodiment, theone or more primer pairs may be selected from: (1) a first primer pairwhich is complementary to a segment of a marker gene specific forStaphylococcus aureus containing sequence modified nucleic acids; (2) asecond primer pair which is complementary to a segment of the mecA genecontaining sequence modified nucleic acids; and (3) a third primer pair,one primer of which is complementary to a segment of SCCmec containingsequence modified nucleic acids and the other primer of which iscomplementary to a segment of the orfx gene containing sequence modifiednucleic acids under conditions wherein the primers specificallyhybridize and an amplification product of the sequence-modified nucleicacids is produced. Two or more of the primer pairs may be combined in asingle reaction vessel for multiplex detection of multiplesequence-modified MRSA markers. The methods may further comprisedetecting an amplification product produced by one or more of the primerpairs, thereby detecting MRSA and/or MSSA, if present, in the sample ofconverted nucleic acids.

In another aspect, the present invention provides methods fordetermining if a biological sample from an individual containsmethicillin resistant Staphylococcus aureus (MRSA) or methicillinsensitive Staphylococcus aureus (MSSA), comprising: (a) converting thenon-methylated cytosines present in the nucleic acids contained in thebiological sample, to uracils to produce sequence-modified nucleicacids; (b) bringing the biological sample containing the sequencemodified nucleic acids in contact with: a first primer pair which iscomplementary to a segment of a marker gene specific for Staphylococcusaureus of the sequence modified nucleic acids; a second primer pairwhich is complementary to a segment of the mecA gene of the sequencemodified nucleic acids; and a third primer pair, one primer of which iscomplementary to a segment of SCCmec of the sequence modified nucleicacids and the other primer of which is complementary to a segment of theorfx gene of the sequence modified nucleic acids under conditionswherein the primers specifically hybridize and amplification products ofthe sequence-modified nucleic acids are produced; and (c) identifyingthe modified nucleic acids from Staphylococcus aureus by detecting theamplification product produced by one or more of the primer pairs. Inthe methods, amplification of all three sequence-modified nucleic acidsindicates MRSA in the sample; and amplification of the S. aureusspecific marker gene alone, or integrated SCCmec and the S. aureusspecific marker gene, but not mecA, indicates MSSA in the sample.

In some embodiments, the marker gene specific for Staphylococcus aureusmay be selected from the group consisting of: spa, agr, ssp protease,sir, sodM, cap, coa, alpha hemolysin, gamma hemolysin, femA, Tufsortase, fibrinogen binding protein, clfB, srC, sdrD, sdrE, sdrF, sdrG,sdrH, NAD synthetase, sar, sbi, rpoB, gyrase A, and orfX. In suitableembodiments, the marker gene specific for S. aureus is spa.

In certain embodiments of the methods described herein, the step ofconverting the non-methylated cytosines present in the nucleic acidscontained in the biological sample to uracils is accomplished bycontacting the nucleic acids with an agent (e.g. sodium bisulfite)capable of converting non-methylated cytosines to uracil.

In another aspect, the present invention provides methods of identifyingmethicillin resistant Staphylococcus aureus (MRSA) or methicillinsensitive Staphylococcus aureus (MSSA), if present, in a biologicalsample, comprising (a) bringing the biological sample in contact with: afirst primer pair which is complementary to a marker gene specific forStaphylococcus aureus; a second primer pair which is complementary tothe mecA gene; and a third primer pair, one primer of which iscomplementary to SCCmec and the other primer of which is complementaryto the orfx gene; under conditions wherein the primers specificallyhybridize and amplify the segments of the marker gene, mecA gene, SCCmecand orfx gene; and (b) identifying the MRSA by detecting anamplification product produced by all of the three primer pairs, whereinamplification of all three sequence-modified nucleic acids indicatesMRSA in the sample; and amplification of the S. aureus specific markergene alone, or integrated SCCmec and the S. aureus specific marker gene,but not mecA, indicates MSSA in the sample.

In some embodiments, any of the primers or probes may be degenerate,i.e. a mixture of primers or probes is provided that have a variablesequence at one or more nucleotide residues. The primers may bedegenerate at 1 nucleotide position, 1-2 nucleotide positions, 1-3nucleotide positions, and at 2, 3, 4, 5, 6, 7, 8, 9, 10 or morenucleotide positions.

The biological sample may be brought into contact with one or more ofthe primer pairs separately or simultaneously. Where the contact occurssimultaneously (i.e. multiplexing), one or more of the first primerpair, the second primer pair, and the third primer pair are brought intocontact with the biological sample and with each other to amplify thetarget sequences. Optionally, a internal positive control nucleic acidand a fourth primer pair complementary to the internal positive controlnucleic acid may be added to the amplification mixture.

In some aspects, the present methods use real time PCR to detect theamplification products. In certain embodiments, the detecting may beaccomplished using a labeled oligonucleotide probe for eachamplification product. A quencher may further be associated with thedetectable label which prevents detection of the label prior toamplification of the probe's target. TaqMan® probes are examples of suchprobes. In some embodiments, the probe and one of the primers of theprimer pair may comprise part of the same molecule (e.g. a Scorpion™primer/probe). A Scorpion™ contains a fluorophore associated with aquencher to reduce background fluorescence. Following PCR extension, thesynthesized target region is attached to the same strand as the probe.Upon denaturation, product, the probe portion of the Scorpion™specifically hybridizes to a part of the newly produced PCR product,physically separating the fluorophore from the quencher, therebyproducing a detectable signal.

In certain embodiments, at least one primer of each primer pair in theamplification reaction is labeled with a detectable moiety. Thus,following amplification, the various target segments can be identifiedby using different detectable moieties such as size and/or color. Thedetectable moiety may be a fluorescent dye. In some embodiments,different pairs of primers in a multiplex PCR may be labeled withdifferent distinguishable detectable moieties. Thus, for example, HEXand FAM fluorescent dyes may be present on different primers inmultiplex PCR and associated with the resulting amplicons. In otherembodiments, the forward primer is be labeled with one detectablemoiety, while the reverse primer is labeled with a different detectablemoiety, e.g. FAM dye for a forward primer and HEX dye for a reverseprimer. Use of different detectable moieties is useful fordiscriminating between amplified products which are of the same lengthor are very similar in length. Thus, in certain embodiments, at leasttwo different fluorescent dyes are used to label different primers usedin a single amplification.

Analysis of amplified products from amplification reactions, such asmultiplex PCR, can be performed using an automated DNA analyzer such asan automated DNA sequencer (e.g., ABI PRISM 3100 Genetic Analyzer) whichcan evaluate the amplified products based on size (determined byelectrophoretic mobility) and/or respective fluorescent label. Detectionof amplification products can also be by melting curve analysis.

In certain embodiments of the aspects provided herein, the methodsfurther comprise a nucleic acid extraction step. Various nucleic acidextraction methods are known in the art which can be employed with themethods and compositions provided herein such as lysis methods (such asalkaline lysis), phenol:chloroform and isopropanol precipitation.Nucleic acid extraction kits can also be used. In suitable embodiments,the extraction method is according to QIAamp™ mini blood kit, AgencourtGenfind™, Roche Cobas®, Roche MagnaPur®, or phenol:chloroform extractionusing Eppendorf Phase Lock Gels®.

Oligonucleotides or combinations of oligonucleotides that are useful asprimers or probes in the methods are also provided. Theseoligonucleotides are provided as substantially purified material.

Kits comprising oligonucleotides which may be primers for performingamplifications as described herein also are provided. Kits may furtherinclude oligonucleotides that may be used as probes to detect amplifiednucleic acid. Kits may also include restriction enzymes for digestingnon-target nucleic acid to increase detection of target nucleic acid bythe oligonucleotide primers.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B present data showing the amplification of thesequence-modified top strand of integrated SCCmec from converted(“CONV”) and unconverted (“UN”) DNA derived from a clinical sample. Thex-axis shows the number of PCR cycles and the y-axis shows thenormalized fluorescence intensity. The threshold cycle (Ct) reflects thecycle number at which the fluorescence generated within a reactioncrosses a user-defined threshold (indicated by the horizontal line).

FIG. 2 presents data showing the amplification of the sequence-modifiedtop strand of the spa marker from converted (“CONV”) and unconverted(“UN”) DNA derived from a clinical sample.

FIG. 3 presents data showing the amplification of the sequence-modifiedtop strand of the mecA marker from converted (“CONV”) and unconverted(“UN”) DNA derived from a clinical sample.

FIGS. 4A and 4B present data showing the amplification of thesequence-modified bottom strand of integrated SCCmec from converted(“CONV”) and unconverted (“UN”) DNA derived from a clinical sample.

FIG. 5 presents data showing the amplification of the sequence-modifiedbottom strand of the spa marker from converted (“CONV”) and unconverted(“UN”) DNA derived from a clinical sample.

FIG. 6 presents data showing the amplification of the sequence-modifiedbottom strand of the mecA marker from converted (“CONV”) and unconverted(“UN”) DNA derived from a clinical sample.

FIG. 7 presents data showing the multiplex amplification thesequence-modified integrated SCCmec, spa, and mecA from converted(“CONV”) and unconverted (“UN”) DNA derived from a clinical sample.

FIG. 8 presents data showing the amplification of the spa marker from aclinical sample.

FIG. 9 presents data showing the amplification of the mecA marker from aclinical sample.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided methods foridentifying methicillin-resistant S. aureus (MRSA) and/ormethicillin-sensitive S. aureus (MSSA) in a biological sample containingnucleic acids by detection of a S. aureus-specific gene, the mecA gene,and integrated SCCmec DNA in the same sample.

In one aspect, the present invention provides methods of identifying S.aureus (MRSA and/or MSSA) in biological samples that may contain nucleicacids from both S. aureus and coagulase-negative Staphylococcal speciessuch as S. epidermidis and S. haemolyticus. Coagulase-negativeStaphylococcal species are less pathogenic than S. aureus but share thesame habitats and permanently or transiently colonize the anterior naresand regions of skin and mucous membranes that are sources of infection.While not wishing to be limited by theory, a single gene marker may beinsufficient to distinguish MRSA from these other less pathogenicstrains. For example, mecA is distributed widely among Staphylococcalstrains, while the SCCmec cassette carrying mecA is known to integrateinto the genomes of S. aureus, S. epidermidis, S. haemolyticus and S.hominis. Species other than S. aureus such as S. epidermidis lackadditional pathogenic factors, making its identification less clinicallysignificant. Hanssen & Sollid. Antimicrob Agents & Chemother 51:1671(2007).

Moreover, the integrated SCCmec cassette can undergo geneticrearrangement, which leaves the SCCmec/orfX junction intact, but deletesthe mecA gene from the genome. Thus, clinical isolates ofmethicillin-sensitive S. aureus (MSSA) exist that contain remnantportions of the SCCmec cassette lacking an intact mecA gene. These MSSAisolates would be falsely identified as MRSA using identificationmethods that solely rely upon detection of the SCCmec cassetteintegrated into the S. aureus genome. For this reason, it is desirablethat a diagnostic assay detects not only the presence of the SCCmeccassette integrated into the S. aureus genome, but also detects thepresence of the mecA gene.

Accordingly, the present inventors have surprisingly discovered that apositive identification of MRSA and/or MSSA can be made by detectingthree marker nucleic acids in a biological sample. To make a positiveidentification of MRSA and/or MSSA, the biological sample containingconverted nucleic acids is contacted with primer pairs corresponding toa S. aureus specific gene (e.g. spa), mecA, and integrated SCCmec. Theamplification preferably occurs in a multiplex format, but individualreactions for each marker may also be used. Amplification from all threesequence-modified markers indicates a high likelihood of MRSA in thesample. Amplification from the S. aureus specific marker alone, orintegrated SCCmec and the S. aureus specific gene, but not mecA,indicates that MSSA is likely present in the sample. In accordance withthe present invention, methods which distinguish between MRSA and MSSAby detecting mecA, integrated SCCmec, and a S. aureus specific markergene may use converted (i.e. sequence-modified) nucleic acids or may useunconverted nucleic acids for any or all of the three genes.

The present invention is described herein using several definitions, asset forth below and throughout the specification.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to“an oligonucleotide” includes a plurality of oligonucleotide molecules,and a reference to “a nucleic acid” is a reference to one or morenucleic acids.

As used herein, “about” means plus or minus 10%.

The terms “amplification” or “amplify” as used herein includes methodsfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. A target nucleic acid may be either DNA or RNA.The sequences amplified in this manner form an “amplicon.” While theexemplary methods described hereinafter relate to amplification usingthe polymerase chain reaction (PCR), numerous other methods are known inthe art for amplification of nucleic acids (e.g., isothermal methods,rolling circle methods, etc.). The skilled artisan will understand thatthese other methods may be used either in place of, or together with,PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” in PCRProtocols, Innis et al., Eds., Academic Press, San Diego, Calif. 1990,pp 13-20; Wharam, et al., Nucleic Acids Res. 2001 Jun. 1;29(11):E54-E54; Hafner, et al., Biotechniques 2001 April; 30(4):852-860.

The term “complement,” “complementary,” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) refersto standard Watson/Crick pairing rules. The complement of a nucleic acidsequence such that the 5′ end of one sequence is paired with the 3′ endof the other, is in “antiparallel association.” For example, thesequence “5′-A-G-T-3′” is complementary to the sequence “3′-T-C-A-5′.”Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids described herein; these include, forexample, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), andPeptide Nucleic Acids (PNA). Complementarity need not be perfect; stableduplexes may contain mismatched base pairs, degenerative, or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs. A complement sequence can also be asequence of RNA complementary to the DNA sequence or its complementsequence, and can also be a cDNA. The term “substantially complementary”as used herein means that two sequences specifically hybridize (definedbelow). The skilled artisan will understand that substantiallycomplementary sequences need not hybridize along their entire length.

As used herein, the term “detecting” used in context of detecting asignal from a detectable label to indicate the presence of a targetnucleic acid in the sample does not require the method to provide 100%sensitivity and/or 100% specificity. As is well known, “sensitivity” isthe probability that a test is positive, given that the person has atarget nucleic acid sequence, while “specificity” is the probabilitythat a test is negative, given that the person does not have the targetnucleic acid sequence. A sensitivity of at least 50% is preferred,although sensitivities of at least 60%, at least 70%, at least 80%, atleast 90% and at least 99% are clearly more preferred. A specificity ofat least 50% is preferred, although sensitivities of at least 60%, atleast 70%, at least 80%, at least 90% and at least 99% are clearly morepreferred. Detecting also encompasses assays with false positives andfalse negatives. False negative rates may be 1%, 5%, 10%, 15%, 20% oreven higher. False positive rates may be 1%, 5%, 10%, 15%, 20% or evenhigher.

A “fragment” in the context of a nucleic acid refers to a sequence ofnucleotide residues which are at least about 5 nucleotides, at leastabout 7 nucleotides, at least about 9 nucleotides, at least about 11nucleotides, or at least about 17 nucleotides. The fragment is typicallyless than about 300 nucleotides, less than about 100 nucleotides, lessthan about 75 nucleotides, less than about 50 nucleotides, or less than30 nucleotides. In certain embodiments, the fragments can be used inpolymerase chain reaction (PCR), various hybridization procedures ormicroarray procedures to identify or amplify identical or related partsof mRNA or DNA molecules. A fragment or segment may uniquely identifyeach polynucleotide sequence of the present invention.

“Genomic nucleic acid” or “genomic DNA” refers to some or all of the DNAfrom a chromosome. Genomic DNA may be intact or fragmented (e.g.,digested with restriction endonucleases by methods known in the art). Insome embodiments, genomic DNA may include sequence from all or a portionof a single gene or from multiple genes. In contrast, the term “totalgenomic nucleic acid” is used herein to refer to the full complement ofDNA contained in the genome. Methods of purifying DNA and/or RNA from avariety of samples are well-known in the art.

The term “multiplex PCR” as used herein refers to simultaneousamplification of two or more products within the same reaction vessel.Each product is primed using a distinct primer pair. A multiplexreaction may further include specific probes for each product, that aredetectably labeled with different detectable moieties.

As used herein, the term “oligonucleotide” refers to a short polymercomposed of deoxyribonucleotides, ribonucleotides or any combinationthereof. Oligonucleotides are generally between about 10, 11, 12, 13, 14or 15 to about 150 nucleotides (nt) in length, more preferably about 10,11, 12, 13, 14, or 15 to about 70 nt, and most preferably between about18 to about 26 nt in length. The single letter code for nucleotides isas described in the U.S. Patent Office Manual of Patent ExaminingProcedure, section 2422, table 1. In this regard, the nucleotidedesignation “R” means purine such as guanine or adenine, “Y” meanspyrimidine such as cytosine or thymidine (uracil if RNA); and “M” meansadenine or cytosine. An oligonucleotide may be used as a primer or as aprobe.

As used herein, a “primer” for amplification is an oligonucleotide thatis complementary to a target nucleotide sequence and leads to additionof nucleotides to the 3′ end of the primer in the presence of a DNA orRNA polymerase. The 3′ nucleotide of the primer should generally beidentical to the target sequence at a corresponding nucleotide positionfor optimal expression and amplification. The term “primer” as usedherein includes all forms of primers that may be synthesized includingpeptide nucleic acid primers, locked nucleic acid primers,phosphorothioate modified primers, labeled primers, and the like. Asused herein, a “forward primer” is a primer that is complementary to theanti-sense strand of dsDNA. A “reverse primer” is complementary to thesense-strand of dsDNA.

Primers are typically between about 10 and about 100 nucleotides inlength, preferably between about 15 and about 60 nucleotides in length,and most preferably between about 25 and about 40 nucleotides in length.There is no standard length for optimal hybridization or polymerasechain reaction amplification. An optimal length for a particular primerapplication may be readily determined in the manner described in H.Erlich, PCR Technology, Principles and Application for DNAAmplification, (1989).

An oligonucleotide (e.g., a probe or a primer) that is specific for atarget nucleic acid will “hybridize” to the target nucleic acid undersuitable conditions. As used herein, “hybridization” or “hybridizing”refers to the process by which an oligonucleotide single strand annealswith a complementary strand through base pairing under definedhybridization conditions.

“Specific hybridization” is an indication that two nucleic acidsequences share a high degree of complementarity. Specific hybridizationcomplexes form under permissive annealing conditions and remainhybridized after any subsequent washing steps. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may occur, for example, at 65° C. in thepresence of about 6×SSC. Stringency of hybridization may be expressed,in part, with reference to the temperature under which the wash stepsare carried out. Such temperatures are typically selected to be about 5°C. to 20° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Equations forcalculating Tm and conditions for nucleic acid hybridization are knownin the art.

As used herein, an oligonucleotide is “specific” for a nucleic acid ifthe oligonucleotide has at least 50% sequence identity with a portion ofthe nucleic acid when the oligonucleotide and the nucleic acid arealigned. An oligonucleotide that is specific for a nucleic acid is onethat, under the appropriate hybridization or washing conditions, iscapable of hybridizing to the target of interest and not substantiallyhybridizing to nucleic acids which are not of interest. Higher levels ofsequence identity are preferred and include at least 75%, at least 80%,at least 85%, at least 90%, at least 95% and more preferably at least98% sequence identity. Sequence identity can be determined using acommercially available computer program with a default setting thatemploys algorithms well known in the art. As used herein, sequences thathave “high sequence identity” have identical nucleotides at least atabout 50% of aligned nucleotide positions, preferably at least at about60% of aligned nucleotide positions, and more preferably at least atabout 75% of aligned nucleotide positions.

Oligonucleotides used as primers or probes for specifically amplifying(i.e., amplifying a particular target nucleic acid sequence) orspecifically detecting (i.e., detecting a particular target nucleic acidsequence) a target nucleic acid generally are capable of specificallyhybridizing to the target nucleic acid.

As used herein, the term “sample” or “test sample” may comprise clinicalsamples, isolated nucleic acids, or isolated microorganisms. Inpreferred embodiments, a sample is obtained from a biological source(i.e., a “biological sample”), such as tissue, bodily fluid, ormicroorganisms collected from a subject. Sample sources include, but arenot limited to, sputum (processed or unprocessed), bronchial alveolarlavage (BAL), bronchial wash (BW), blood, bodily fluids, cerebrospinalfluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material).Preferred sample sources include nasopharyngeal swabs, wound swabs, andnasal washes. The term “patient sample” as used herein refers to asample obtained from a human seeking diagnosis and/or treatment of adisease.

As used herein, the term “Scorpion™ detection system” refers to a methodfor real-time PCR. This method utilizes a bi-functional molecule(referred to herein as a “Scorpion™”), which contains a PCR primerelement covalently linked by a polymerase-blocking group to a probeelement. Additionally, each Scorpion™ molecule contains a fluorophorethat interacts with a quencher to reduce the background fluorescence.

The terms “target nucleic acid” or “target sequence” as used hereinrefer to a sequence which includes a segment of nucleotides of interestto be amplified and detected. Copies of the target sequence which aregenerated during the amplification reaction are referred to asamplification products, amplimers, or amplicons. Target nucleic acid maybe composed of segments of a chromosome, a complete gene with or withoutintergenic sequence, segments or portions of a gene with or withoutintergenic sequence, or sequence of nucleic acids which probes orprimers are designed. Target nucleic acids may include a wild-typesequence(s), a mutation, deletion or duplication, tandem repeat regions,a gene of interest, a region of a gene of interest or any upstream ordownstream region thereof. Target nucleic acids may representalternative sequences or alleles of a particular gene. Target nucleicacids may be derived from genomic DNA, cDNA, or RNA. As used hereintarget nucleic acid may be DNA or RNA extracted from a cell or a nucleicacid copied or amplified therefrom, or may include extracted nucleicacids further converted using a bisulfite reaction.

As used herein “TaqMan® PCR detection system” refers to a method forreal time PCR. In this method, a TaqMan® probe which hybridizes to thenucleic acid segment amplified is included in the PCR reaction mix. TheTaqMan® probe comprises a donor and a quencher fluorophore on either endof the probe and in close enough proximity to each other so that thefluorescence of the donor is taken up by the quencher. However, when theprobe hybridizes to the amplified segment, the 5′-exonuclease activityof the Taq polymerase cleaves the probe thereby allowing the donorfluorophore to emit fluorescence which can be detected.

Sample Preparation

Specimens from which MRSA can be detected and quantified with thepresent invention are from sterile and/or non-sterile sites. Sterilesites from which specimens can be taken are body fluids such as blood,urine, cerebrospinal fluid, synovial fluid, pleural fluid, pericardialfluid, intraocular fluid, tissue biopsies or endotracheal aspirates.Non-sterile sites from which specimens can be taken are e.g. sputum,stool, swabs from e.g. skin, inguinal, nasal and/or throat. Preferably,specimens are from non-sterile sites, more preferably wound and/or nasalswabs are used in the present invention. Specimens for MRSA detectionmay also comprise cultures of isolated bacteria grown on appropriatemedia to form colonies. Specimens may also include bacterial isolates.

Specimens may be processed prior to nucleic acid amplification. In oneembodiment, bacteria isolated from clinical specimens may be cultured inmedia containing antibiotics (e.g. methicillin) to check for thepresence of drug resistance. In another embodiment, immunocapture withan antibody specific for S. aureus is used to enrich the sample for thisspecies. To discriminate MRSA from any other methicillin-resistantStaphylococcal species, the assay first detects a species-specific geneproduct, e.g. spa., using an antibody, and then uses nucleic acidamplification to detect one or more target nucleic acids associated withMRSA (e.g. mecA and/or SCCmec) in the enriched sample. In anotherembodiment, capture of S. aureus genomic DNA using a specific bindingagent, such as a nucleic acid probe or protein nucleic acid, is used toenrich the sample for this species. For example, the assay first detectsa species-specific gene product e.g. spa using a nucleic acid probe, andthen uses nucleic acid amplification to detect one or more targetnucleic acids associated with MRSA (e.g. mecA and/or SCCmec) in theenriched sample. The nucleic acid conversion step may be done before orafter the genomic capture step.

The nucleic acid (DNA or RNA) may be isolated from the sample accordingto any methods well known to those of skill in the art. If necessary thesample may be collected or concentrated by centrifugation and the like.The cells of the sample may be subjected to lysis, such as by treatmentswith enzymes, heat surfactants, ultrasonication or combination thereof.The lysis treatment is performed in order to obtain a sufficient amountof DNA derived from MRSA and/or MSSA, if present in the sample, todetect using polymerase chain reaction.

Various methods of DNA extraction are suitable for isolating the DNA.Suitable methods include phenol and chloroform extraction. See Maniatiset al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring HarborLaboratory Press, page 16.54 (1989). Numerous commercial kits also yieldsuitable DNA including, but not limited to, QIAamp™ mini blood kit,Agencourt Genfind™, Roche Cobas® Roche MagNA Pure® or phenol:chloroformextraction using Eppendorf Phase Lock Gels®.

Conversion of Nucleic Acids to Modified Nucleic Acids

In one aspect, the nucleic acids present in the sample are converted tosequence-modified nucleic acids prior to amplification. “Conversion”refers to the process whereby the non-methylated cytosines present inthe nucleic acids are chemically deaminated and modified into uracils.Following amplification, thymidine residues are substituted for thedeaminated cytosines. In some methods, the conversion is accomplished bycontacting the nucleic acids with sodium bisulphite. Thus, inunmethylated DNA, this process results in all or mostly all cystosine(C) residues being replaced by thymidine (T), thereby converting a 4base pair sequence into a 3 base pair sequence of A's, T's and G's. Thebisulphite DNA conversion method for the detection of methylated DNA wasdescribed in Frommer et al., PNAS 89: 1827-1831 (1992) and Clark et al.,Nucl Acids Res 22: 2990-7 (1994). Numerous commercial kits are availableto perform the bisulfite conversion reaction including MethylEasy™(Human Genetic Signatures), EpiTect® Bisulfite Kit (Qiagen/Epigenomics),and MethylAmp™ DNA Modification Kit (Epigentek).

Chemical conversion of cytosine to thymidine residues may be carried outas follows. First, the nucleic acid sample is denatured, if doublestranded, to provide single-stranded nucleic acid. The denaturation stepmay be performed by contacting the nucleic acid with a NaOH solution, orother suitable alkaline reagent, or by heating. Second, the nucleic acidsample is reacted with a reagent and incubated so as to form a treatednucleic acid sample where any methylated nucleotides in the nucleic acidsample remain unchanged while unmethylated cytosine nucleotides aredepurinated. Suitable reagents include, but are not limited to, sodiumbisulfite. Third, the treated nucleic acid sample is purified tosubstantially remove any unwanted reagents or diluents from the treatednucleic acid sample. This may be accomplished, for example, by usingcolumn purification and concentration, or diluting the sample so as toreduce salt concentration and then precipitating the nucleic acid.Finally, a desulphonation step of the treated nucleic acid sample may beperformed to remove sulphonate groups present on the treated nucleicacid so as to obtain a nucleic acid sample substantially free ofsulphonate groups. Further detail regarding the conversion ofnon-methylated nucleotides can be found in U.S. Patent Applicationpublications 2007/0020633, 2004/0219539, and 2004/0086944.

Non-methylated cytosine residues in both DNA strands are converted as aresult of the process just described. Consequently, following conversionthe two DNA strands are no longer fully complementary and will notspecifically hybridize, but may hybridize under non-stringentconditions, depending on the number of non-methylated cytosines withinthe converted strands. If few non-methylated cytosines are presentwithin the strand, then the strands will likely retain somecomplementarity after conversion. If many non-methylated cytosines arepresent within the strand, then the top strand and bottom strand will beless likely to hybridize even under non-stringent conditions. As usedherein, the general term “strand” refers to a single chain ofsugar-phosphate linked nucleosides, i.e. a strand of double-stranded DNA(dsDNA). The “top strand” refers to the sense strand of thepolynucleotide read in the 5′ to 3′ direction, which is the strand ofdsDNA that includes at least a portion of a coding sequence of afunctional protein. The “bottom strand” refers to the anti-sense strand,which is the strand of dsDNA that is the reverse complement of the sensestrand. It is understood that, while a sequence is referred to as bottomor top strand, such a designation is intended to distinguishcomplementary strands since, in solution, there is no orientation thatfixes a strand as a top or bottom strand. It is also understood that thetop strand will therefore have its own complementary strand followingamplification and likewise the bottom strand will have its owncomplementary strand following amplification. While the originalconverted strands (top or bottom) will be simplified to only contain a 3base pair sequence of A's, T's and G's, the complementary strands willnecessarily only contain T's, A's and C's. In some methods, the presenceof converted nucleic acids is detected using PCR. For each targetsequence, either the top strand, the bottom strand, or both may bedetected using primers specific for the modified sequence of eitherstrand.

Amplification of Nucleic Acids

Nucleic acid samples or isolated nucleic acids may be amplified byvarious methods known to the skilled artisan. Preferably, PCR is used toamplify nucleic acids of interest. Briefly, in PCR, two primer sequencesare prepared that are complementary to regions on opposite complementarystrands of the marker sequence. An excess of deoxynucleotidetriphosphates are added to a reaction mixture along with a DNApolymerase, e.g., Taq polymerase. When the template issequence-modified, as described above, the amplification mixturepreferably does not contain a UNG nuclease.

If the target sequence is present in a sample, the primers will bind tothe sequence and the polymerase will cause the primers to be extendedalong the target sequence by adding on nucleotides. By raising andlowering the temperature of the reaction mixture, the extended primerswill dissociate from the marker to form reaction products, excessprimers will bind to the marker and to the reaction products and theprocess is repeated, thereby generating amplification products. Cyclingparameters can be varied, depending on the length of the amplificationproducts to be extended. An internal positive amplification control(IPC) can be included in the sample, utilizing oligonucleotide primersand/or probes. The IPC can be used to monitor both the conversionprocess and any subsequent amplification.

Target Nucleic Acids and Primers

In various embodiments of the present invention, oligonucleotide primersand probes are used in the methods described herein to amplify anddetect target sequence-modified nucleic acids specific to MRSA and/orMSSA. In certain embodiments, target nucleic acids may includesequence-modified fragments of the mecA gene, integrated SCCmec, and amarker gene specific to Staphylococcus aureus (e.g. spa). In otherembodiments, target nucleic acids may include unmodified fragments ofthe mecA gene, integrated SCCmec, and a marker gene specific toStaphylococcus aureus (e.g. spa). In addition, primers can also be usedto amplify one or more control nucleic acid sequences. The targetnucleic acids described herein may be detected individually or in amultiplex format, utilizing individual labels for each target.

The skilled artisan is capable of designing and preparing primers thatare appropriate for amplifying a target sequence in view of thisdisclosure. The length of the amplification primers for use in thepresent invention depends on several factors including the nucleotidesequence identity and the temperature at which these nucleic acids arehybridized or used during in vitro nucleic acid amplification. Theconsiderations necessary to determine a preferred length for anamplification primer of a particular sequence identity are well known tothe person of ordinary skill in the art.

Specifically, primers and probes to amplify and detect sequence-modifiedor unmodified nucleic acids corresponding to integrated SCCmec, mecA,and a marker specific to Staphylococcus aureus (e.g. spa) are providedby the invention. Primers that amplify a nucleic acid molecule can bedesigned using, for example, a computer program such as OLIGO (MolecularBiology Insights, Inc., Cascade, Colo.). Important features whendesigning oligonucleotides to be used as amplification primers include,but are not limited to, an appropriate size amplification product tofacilitate detection (e.g., by electrophoresis or real-time PCR),similar melting temperatures for the members of a pair of primers, andthe length of each primer (i.e., the primers need to be long enough toanneal with sequence-specificity and to initiate synthesis but not solong that fidelity is reduced during oligonucleotide synthesis).Typically, oligonucleotide primers are 15 to 35 nucleotides in length.

A further consideration for designing primers for sequence-modifiednucleic acids is that the converted sequence comprises primarily A, T,and G residues or alternatively primarily T, A and C residues.Accordingly, the melting temperature of the primer directed to asequence-modified target will typically be lower than a correspondingprimer directed to the unmodified target. Therefore, it may be necessaryfor the length of sequence-modified primers to be adjusted compared to acorresponding unmodified target primer. Therefore, the oligonucleotideprimers may be longer than typical oligonucleotide primers directed tosequences comprised of all four bases (e.g., longer than 15 to 35nucleotides). When the PCR template is sequence modified DNA, themajority of the DNA is effectively reduced to three bases (A, T, and Gon one strand and T, A and C on the other strand). This decreases thecomplexity of DNA and can increase the incidence of primer-templateinteraction at “non-specific” regions. Optionally, these non-specificinteractions may be overcome by the use of a nested or semi-nested PCRapproach.

Designing oligonucleotides to be used as hybridization probes can beperformed in a manner similar to the design of primers. As witholigonucleotide primers, oligonucleotide probes usually have similarmelting temperatures, and the length of each probe must be sufficientfor sequence-specific hybridization to occur but not so long thatfidelity is reduced during synthesis. Oligonucleotide probes aregenerally 15 to 60 nucleotides in length.

In some embodiments, a mix of primers is provided having degeneracy atone or more nucleotide positions. Degenerate primers are used in PCRwhere variability exists in the target sequence, i.e. the sequenceinformation is ambiguous. Typically, degenerate primers will exhibitvariability at no more than about 4, no more than about 3, preferably nomore than about 2, and most preferably, no more than about 1 nucleotideposition.

The target nucleic acids to identify MRSA may be selected according to awide variety of methods. Exemplary target nucleic acids are modifiedsequences corresponding to mecA integrated SCCmec, and a marker specificto Staphylococcus aureus (e.g. spa). The target may be amplified infull. Alternatively, in some embodiments, fragments or segments of thetarget sequences are amplified. The fragment may be derived from anyregion of the full sequence, but fragment length in accordance with thepresent methods is typically at least 30, at least 50, at least 75, atleast 100, at least 150, at least 200, at least 250 or at least 300nucleotides. As will be understood by one of skill in the art, the sizeand location of the particular target nucleic acid will control theselection of the amplification primers and vice versa.

In some embodiments, specific primers and probes are selected to amplifyand detect a modified fragment of a marker gene specific for S. aureus.This marker should be present in S. aureus, but absent from otherStaphylococcus species. Examples of specific marker genes include, butare not limited to spa, agr, ssp protease, sir, sodM, cap, coa, alphahemolysin, gamma hemolysin, femA, Tuf; sortase, fibrinogen bindingprotein, clfB, srC, sdrD, sdrE, sdrF, sdrG, sdrH, NAD synthetase, sar,sbi, rpoB, gyrase A, and orfX. The detection of a S. aureus-specificgene helps to distinguish a sample containing S. aureus from one thatmay contain other less pathogenic species or strains, e.g. S.epidermidis. Thus, amplification of a sequence-modified S.aureus-specific gene, together with sequence-modified mecA andsequence-modified integrated SCCmec distinguishes between MRSA andmethicillin-resistant S. epidermidis (MRSE) and MSSA. One suitablemarker gene is the 1.55 kb spa gene (see, for example, GenBank AccessionNo. NC_(—)002952, range 125378-123828). Exemplary primer/Scorpion™sequences for amplifying and detecting sequence-modified spa include:SEQ ID NOS:10-11 and 25-26. The skilled artisan will understand thatother primers, probes, and Scorpions™ may be used.

In some embodiments, specific primers and probes are selected to amplifyand detect a fragment of the 2.0 kb mecA gene (see, for example, GenBankAccession No. AB033763) or a fragment of the sequence-modified mecAgene. Exemplary primer/Scorpion™ sequences for amplifying and detectingsequence-modified mecA include: SEQ ID NOS:12-13, and 27-28. The skilledartisan will understand that other primers, probes, and Scorpions™ maybe used.

In some embodiments, specific primers and probes are selected to amplifyand detect a fragment of the integrated SCCmec or sequence-modifiedintegrated SCCmec cassette. To detect this sequence, primers aredesigned so that the amplified fragment contains the junction betweenthe SCCmec cassette and the surrounding genomic DNA. The primers may bedesigned to amplify either the 5′ or 3′ junction of sequence-modifiedSCCmec integrated within the S. aureus genome. Preferably the 3′junction of sequence-modified SCCmec is amplified. In particular, aforward primer may be designed to specifically hybridize to the 3′ endof sequence-modified SCCmec and a reverse primer designed tospecifically hybridize to the sequence-modified orfX gene in thechromosomal DNA surrounding the sequence-modified SCCmec. Exemplaryprimer/Scorpion™ sequences for amplifying and detectingsequence-modified integrated SCCmec include: SEQ ID NOS:1-9, and 14-24.The skilled artisan will understand that other primers, probes, andScorpions™ may be used.

In a suitable embodiment, PCR is performed using a Scorpion™primer/probe combination. Scorpion™ probes, as used in the presentinvention comprise a 3′ primer with a 5′ extended probe tail comprisinga hairpin structure which possesses a fluorophore/quencher pair. DuringPCR, the polymerase is blocked from extending into the probe tail by theinclusion of hexethlyene glycol (HEG). During the first round ofamplification the 3′ target-specific primer anneals to the target and isextended such that the Scorpion™ is now incorporated into the newlysynthesized strand, which possesses a newly synthesized target regionfor the 5′ probe. During the next round of denaturation and annealing,the probe region of the Scorpion™ hairpin loop will hybridize to thetarget, thus separating the fluorophore and quencher and creating ameasurable signal. Such probes are described in Whitcombe et al., NatureBiotech 17: 804-807 (1999).

Detection of a Amplified Nucleic Acids

Amplification of nucleic acids can be detected by any of a number ofmethods well-known in the art such as gel electrophoresis, columnchromatography, hybridization with a probe, sequencing, melting curveanalysis, or “real-time” detection.

In one approach, sequences from two or more fragments of interest areamplified in the same reaction vessel (i.e. “multiplex PCR”). Detectioncan take place by measuring the end-point of the reaction or in “realtime.” For real-time detection, primers and/or probes may be detectablylabeled to allow differences in fluorescence when the primers becomeincorporated or when the probes are hybridized, for example, andamplified in an instrument capable of monitoring the change influorescence during the reaction. Real-time detection methods fornucleic acid amplification are well known and include, for example, theTaqMan® system, Scorpion™ primer system and use of intercalating dyesfor double stranded nucleic acid.

In end-point detection, the amplicon(s) could be detected by firstsize-separating the amplicons, then detecting the size-separatedamplicons. The separation of amplicons of different sizes can beaccomplished by, for example, gel electrophoresis, columnchromatography, or capillary electrophoresis. These and other separationmethods are well-known in the art. In one example, amplicons of about 10to about 150 base pairs whose sizes differ by 10 or more base pairs canbe separated, for example, on a 4% to 5% agarose gel (a 2% to 3% agarosegel for about 150 to about 300 base pair amplicons), or a 6% to 10%polyacrylamide gel. The separated nucleic acids can then be stained witha dye such as ethidium bromide and the size of the resulting stainedband or bands can be compared to a standard DNA ladder.

In another example, Invader™ (Third Wave Technologies, Inc.) may be usedto detect specific nucleic acid sequences after linear or exponentialamplification. In the Invader™ assay, the DNA structure recognized by athermostable flap endonuclease (FEN), is formed by an Invader™ probethat overlaps the signal probe by at least one base. The unpairedsingle-stranded flap of the signal probe is released during the FENreaction and can be detected by various methods such as measuringfluorescence after capturing and extending the released signal probeflap with fluorescein-labeled nucleotides (ELISA-format),mass-spectrometry, denaturing gel electrophoresis, etc. A variation ofthe Invader™ assay uses a FRET probe. The released signal probe fragmentof the initial FEN reaction subsequently serves as an Invader probeinvading the stem fragment of the hairpin formed intramolecularly in theFRET probe. This process induces a second FEN reaction during which thefluorophore in the FRET probe is separated from the nearby quenching dyein the FRET probe, resulting in the generation of fluorescence. Both FENreactions occur at isothermic conditions (near the melting temperatureof the probes) which enables a linear signal amplification.

In another embodiment, two or more fragments of interest are amplifiedin separate reaction vessels. If the amplification is specific, that is,one primer pair amplifies for one fragment of interest but not theother, detection of amplification is sufficient to distinguish betweenthe two types—size separation would not be required.

In some embodiments, amplified nucleic acids are detected byhybridization with a specific probe. Probe oligonucleotides,complementary to a portion of the amplified target sequence may be usedto detect amplified fragments. Hybridization may be detected in realtime or in non-real time. Amplified nucleic acids for each of the targetsequences may be detected simultaneously (i.e., in the same reactionvessel) or individually (i.e., in separate reaction vessels). Inpreferred embodiments, the amplified DNA is detected simultaneously,using two or more distinguishably-labeled, gene-specific oligonucleotideprobes, one which hybridizes to the first target sequence and one whichhybridizes to the second target sequence. For sequence-modified nucleicacids, the target may be independently selected from the top strand orthe bottom strand. Thus, all targets to be detected may comprise topstrand, bottom strand, or a combination of top strand and bottom strandtargets.

The probe may be detectably labeled by methods known in the art. Usefullabels include, e.g., fluorescent dyes (e.g., Cy5®, Cy3®, FITC,rhodamine, lanthamide phosphors, Texas red, FAM, JOE, Cal Fluor Red610®, Quasar 670®), ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I, electron-densereagents (e.g., gold), enzymes, e.g., as commonly used in an ELISA(e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkalinephosphatase), colorimetric labels (e.g., colloidal gold), magneticlabels (e.g., Dynabeads™), biotin, dioxigenin, or haptens and proteinsfor which antisera or monoclonal antibodies are available. Other labelsinclude ligands or oligonucleotides capable of forming a complex withthe corresponding receptor or oligonucleotide complement, respectively.The label can be directly incorporated into the nucleic acid to bedetected, or it can be attached to a probe (e.g., an oligonucleotide) orantibody that hybridizes or binds to the nucleic acid to be detected.

One general method for real time PCR uses fluorescent probes such as theTaqMan® probes, molecular beacons, and Scorpions. Real-time PCRquantitates the initial amount of the template with more specificity,sensitivity and reproducibility, than other forms of quantitative PCR,which detect the amount of final amplified product. Real-time PCR doesnot detect the size of the amplicon. The probes employed in Scorpion™and TaqMan® technologies are based on the principle of fluorescencequenching and involve a donor fluorophore and a quenching moiety.

In a preferred embodiment, the detectable label is a fluorophore. Theterm “fluorophore” as used herein refers to a molecule that absorbslight at a particular wavelength (excitation frequency) and subsequentlyemits light of a longer wavelength (emission frequency). The term “donorfluorophore” as used herein means a fluorophore that, when in closeproximity to a quencher moiety, donates or transfers emission energy tothe quencher. As a result of donating energy to the quencher moiety, thedonor fluorophore will itself emit less light at a particular emissionfrequency that it would have in the absence of a closely positionedquencher moiety.

The term “quencher moiety” as used herein means a molecule that, inclose proximity to a donor fluorophore, takes up emission energygenerated by the donor and either dissipates the energy as heat or emitslight of a longer wavelength than the emission wavelength of the donor.In the latter case, the quencher is considered to be an acceptorfluorophore. The quenching moiety can act via proximal (i.e.,collisional) quenching or by Förster or fluorescence resonance energytransfer (“FRET”). Quenching by FRET is generally used in TaqMan® probeswhile proximal quenching is used in molecular beacon and Scorpion™ typeprobes.

In proximal quenching (a.k.a. “contact” or “collisional” quenching), thedonor is in close proximity to the quencher moiety such that energy ofthe donor is transferred to the quencher, which dissipates the energy asheat as opposed to a fluorescence emission. In FRET quenching, the donorfluorophore transfers its energy to a quencher which releases the energyas fluorescence at a higher wavelength. Proximal quenching requires veryclose positioning of the donor and quencher moiety, while FRETquenching, also distance related, occurs over a greater distance(generally 1-10 nm, the energy transfer depending on R-6, where R is thedistance between the donor and the acceptor). Thus, when FRET quenchingis involved, the quenching moiety is an acceptor fluorophore that has anexcitation frequency spectrum that overlaps with the donor emissionfrequency spectrum. When quenching by FRET is employed, the assay maydetect an increase in donor fluorophore fluorescence resulting fromincreased distance between the donor and the quencher (acceptorfluorophore) or a decrease in acceptor fluorophore emission resultingfrom decreased distance between the donor and the quencher (acceptorfluorophore).

Suitable fluorescent moieties include the following fluorophores knownin the art: 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid,acridine and derivatives (acridine, acridine isothiocyanate) AlexaFluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, AlexaFluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes),5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate(Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide,Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies), BODIPY® R-6G,BOPIPY®R 530/550, BODIPY® FL, Brilliant Yellow, coumarin and derivatives(coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumarin 151)), Cy2®, Cy3®, Cy3.5®,Cy5®, Cy5.5®, cyanosine, 4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin,diethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), Eclipse™(Epoch Biosciences Inc.), eosin and derivatives (eosin, eosinisothiocyanate), erythrosin and derivatives (erythrosin B, erythrosinisothiocyanate), ethidium, fluorescein and derivatives(5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein(HEX), QFITC (XRITC), tetrachlorofluorescein (TET)), fluorescamine,IR144, IR1446, Malachite Green isothiocyanate, 4-methylumbelliferone,ortho cresolphthalein, nitrotyrosine, pararosaniline, Phenol Red,B-phycoerythrin, R-phycoerythrin, o-phthaldialdehyde, Oregon Green®,propidium iodide, pyrene and derivatives (pyrene, pyrene butyrate,succinimidyl 1-pyrene butyrate), QSY® 7, QSY® 9, QSY® 21, QSY® 35(Molecular Probes), Reactive Red 4 (Cibacron® Brilliant Red 3B-A),rhodamine and derivatives (6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamineX isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonylchloride derivative of sulforhodamine 101 (Texas Red)),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid,terbium chelate derivatives.

Other fluorescent nucleotide analogs can be used, see, e.g., Jameson,278 Meth. Enzymol. 363-390 (1997); Zhu, 22 Nucl. Acids Res. 3418-3422(1994). U.S. Pat. Nos. 5,652,099 and 6,268,132 also describe nucleosideanalogs for incorporation into nucleic acids, e.g., DNA and/or RNA, oroligonucleotides, via either enzymatic or chemical synthesis to producefluorescent oligonucleotides. U.S. Pat. No. 5,135,717 describesphthalocyanine and tetrabenztriazaporphyrin reagents for use asfluorescent labels.

The detectable label can be incorporated into, associated with orconjugated to a nucleic acid. Label can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance or impact on otheruseful or desired properties. See, e.g., Mansfield, 9 Mol. Cell. Probes145-156 (1995). Detectable labels can be incorporated into nucleic acidsby covalent or non-covalent means, e.g., by transcription, such as byrandom-primer labeling using Klenow polymerase, or nick translation, oramplification, or equivalent as is known in the art. For example, anucleotide base is conjugated to a detectable moiety, such as afluorescent dye, and then incorporated into nucleic acids during nucleicacid synthesis or amplification.

With Scorpion™ probes, sequence-specific priming and PCR productdetection is achieved using a single molecule. The Scorpion™ probemaintains a stem-loop configuration in the unhybridized state. Thefluorophore is attached to the 5′ end and is quenched by a moietycoupled to the 3′ end, although in suitable embodiments, thisarrangement may be switched The 3′ portion of the stem also containssequence that is complementary to the extension product of the primer.This sequence is linked to the 5′ end of a specific primer via anon-amplifiable monomer. After extension of the Scorpion™ primer, thespecific probe sequence is able to bind to its complement within theextended amplicon thus opening up the hairpin loop. This prevents thefluorescence from being quenched and a signal is observed. A specifictarget is amplified by the reverse primer and the primer portion of theScorpion™, resulting in an extension product. A fluorescent signal isgenerated due to the separation of the fluorophore from the quencherresulting from the binding of the probe element of the Scorpion™ to theextension product.

TaqMan® probes (Heid, et al., Genome Res 6: 986-994, 1996) use thefluorogenic 5′ exonuclease activity of Taq polymerase to measure theamount of target sequences in cDNA samples. TaqMan® probes areoligonucleotides that contain a donor fluorophore usually at or near the5′ base, and a quenching moiety typically at or near the 3′ base. Thequencher moiety may be a dye such as TAMRA or may be a non-fluorescentmolecule such as 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL). SeeTyagi, et al., 16 Nature Biotechnology 49-53 (1998). When irradiated,the excited fluorescent donor transfers energy to the nearby quenchingmoiety by FRET rather than fluorescing. Thus, the close proximity of thedonor and quencher prevents emission of donor fluorescence while theprobe is intact.

TaqMan® probes are designed to anneal to an internal region of a PCRproduct. When the polymerase (e.g., reverse transcriptase) replicates atemplate on which a TaqMan® probe is bound, its 5′ exonuclease activitycleaves the probe. This ends the activity of the quencher (no FRET) andthe donor fluorophore starts to emit fluorescence which increases ineach cycle proportional to the rate of probe cleavage. Accumulation ofPCR product is detected by monitoring the increase in fluorescence ofthe reporter dye (note that primers are not labeled). If the quencher isan acceptor fluorophore, then accumulation of PCR product can bedetected by monitoring the decrease in fluorescence of the acceptorfluorophore.

In a suitable embodiment, real time PCR is performed using any suitableinstrument capable of detecting fluorescence from one or morefluorescent labels. For example, real time detection on the instrument(e.g. a ABI Prisms 7900HT sequence detector) monitors fluorescence andcalculates the measure of reporter signal, or Rn value, during each PCRcycle. The threshold cycle, or Ct value, is the cycle at whichfluorescence intersects the threshold value. The threshold value isdetermined by the sequence detection system software or manually.

In some embodiments, melting curve analysis may be used to detect anamplification product. Melting curve analysis involves determining themelting temperature of an nucleic acid amplicon by exposing the ampliconto a temperature gradient and observing a detectable signal from afluorophore. Melting curve analysis is based on the fact that a nucleicacid sequence melts at a characteristic temperature called the meltingtemperature (Tm), which is defined as the temperature at which half ofthe DNA duplexes have separated into single strands. The meltingtemperature of a DNA depends primarily upon its nucleotide composition.Thus, DNA molecules rich in G and C nucleotides have a higher Tm thanthose having an abundance of A and T nucleotides.

Where a fluorescent dye is used to determine the melting temperature ofa nucleic acid in the method, the fluorescent dye may emit a signal thatcan be distinguished from a signal emitted by any other of the differentfluorescent dyes that are used to label the oligonucleotides. In someembodiments, the fluorescent dye for determining the melting temperatureof a nucleic acid may be excited by different wavelength energy than anyother of the different fluorescent dyes that are used to label theoligonucleotides. In some embodiments, the second fluorescent dye fordetermining the melting temperature of the detected nucleic acid is anintercalating agent. Suitable intercalating agents may include, but arenot limited to SYBR™ Green 1 dye, SYBR dyes, Pico Green, SYTO dyes,SYTOX dyes, ethidium bromide, ethidium homodimer-1, ethidiumhomodimer-2, ethidium derivatives, acridine, acridine orange, acridinederivatives, ethidium-acridine heterodimer, ethidium monoazide,propidium iodide, cyanine monomers, 7-aminoactinomycin D, YOYO-1,TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1,cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5,PO-PRO-1, BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1, and mixturethereof. In suitable embodiments, the selected intercalating agent isSYBR™ Green 1 dye.

By detecting the temperature at which the fluorescence signal is lost,the melting temperature can be determined. In the disclosed methods,each of the amplified target nucleic acids may have different meltingtemperatures. For example, each of these amplified target nucleic acidsmay have a melting temperature that differs by at least about 1° C.,more preferably by at least about 2° C., or even more preferably by atleast about 4° C. from the melting temperature of any of the otheramplified target nucleic acids. The melting temperature(s) of the MRSAtargets from the respective amplification product can confirm thepresence or absence of MRSA and/or MSSA in the sample.

To minimize the potential for cross contamination, reagent and mastermixpreparation, specimen processing and PCR setup, and amplification anddetection are all carried out in physically separated areas.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

EXAMPLE I Conversion of Native Nucleic Acids to Sequence ModifiedNucleic Acids

Genomic DNA was extracted from 6 nasal swab specimens using the QIAamp™mini blood kit (Qiagen) extraction procedure with the ATL buffermodification. Nasal swab specimens were stored at −20° C. prior toextraction. Just prior to extraction, the swabs were thawed at roomtemperature for 20 minutes, 500 μL 1×PBS was added to the swab and thevial containing the swab followed by gentle agitation to mix. 450 μL ofthe suspension was then transferred to a 1.5 ml microcentrifuge tube andthe suspension was centrifuged at 14,000 rpm for 10 min. The supernatantwas carefully aspirated and the pellet was resuspended in 180 μL ofBuffer ATL from the QIAamp™ mini blood kit. This kit was used to extractDNA according to the manufacturer's instructions. Eluted genomic DNA wassplit into two samples: one for conversion of nucleic acids to sequencemodified nucleic acids (“converted DNA”) and the other was not converted(“unconverted DNA”).

Genomic DNA was converted using the MethylEasy™ Fast kit (Human GeneticSignatures). The manufacturer's protocol was followed. At the end of theprocedure, converted DNA was eluted in 2 steps, each using 15 μL reagent5 from the MethylEasy™ Fast kit for a total elution volume of 30 μL.

EXAMPLE 2 Amplification of Sequence Modified Nucleic Acids Top StrandAssays

To detect the sequence-modified top strand of integrated SCCmec, sevenforward primers and two Scorpion™ oligonucleotides containing a reverseorfx primer and probe were designed. These primers detect the top strandof sequence modified integrated SCCmec MREJ (mec right extremejunction), types i & ii, iii, iv, v, and vii (Table 1). The forwardprimers were each specific to a single SCCmec type, but the Scorpion™primer/probe, which hybridizes to orfx was not specific for any singleSCCmec type. One Scorpion™ primer/probe and five forward primers arecombined to detect sequence-modified SCCmec cassette types ii, iii, iv,v, and vii.

To determine the specificity of the primers shown in Table 1 forsequence-modified top strand of integrated SCCmec, samples of “convertedDNA” or “unconverted DNA” were analyzed in singleplex reactions. A 10×Primer Mix was prepared using 0.1×TE pH 8 as the diluent. Finalconcentrations of each component in the 10× mix indicated inparenthesis: SA4-MTopF1.1 (2 μM), SA4-MTop2.1 (2 μM), SA4-MTop3.1, (2μM) SA4-MTopF4 (2 μM), SA4-MTop5 (2 μM), SA4-MSRTop1 (2 μM) orSA4-MSRTop2 (2 μM). The reaction mix for a single well of a 96-welloptical PCR plate included the following: 12.5 μL 2× Master Mix; 2.5 μLof 10× Primer Mix (components listed above); 4.0 μL Nuclease-free water;1.0 μL Internal control amplicon; 5.0 μL “converted DNA” or “unconvertedDNA” template (250 pg). The conditions for the real time PCR run on aApplied Biosystems AB7500 were as follows: Step 1: 95° C. 10 min; Step2: 95° C. 15 sec; Step 3: 60° C. 35 sec. Steps 2-3 were repeated for atotal of 45 cycles. Sequence-modified integrated SCCmec was detectedwith Cal Fluor Red 610.

TABLE 1 Top Strand Assay Scorpions and Primers for Integrated SCCmec TopStrand Scorpion ™ or SCCmec Primer Name MREJ Type (SEQ ID NO) SequenceScorpion ™ SA4-MSRTop1 5′ quencher-AGGCGG (types i, ii, iii, (SEQ IDNO:1) TTGTAAGATGTTTTTGTG iv, v, vii) TAGGTTGTCCGCCTdye/blocker-AAAACAAAAC AACTTTATWTTCATCATT AACA3′ Scorpion ™ SA4-MSRTop25′ quencher-AGGCGG (types i, ii, iii, (SEQ ID NO:2) AATGTTATTTTGTTRAATiv, v, vii) GATAGTGtCCGCCT- dye/blocker-AACAAC CTACACAAAAACATCTTA CAACA3′ i, ii SA4-MTopF1.1 5′ TATTTGAAATGAAAG (SEQ ID NO:3) ATTGTGGAGGTTA 3iii SA4-MTopF2.1 5′ TATGATATTGTAAGG (SEQ ID NO:4) TATAATTTAATATTTTATATATGTA 3′ iii SA4-MTopF2.2 5′ TATTATTAATTTTTT (SEQ ID NO:5)AATTTAATTGTAGTTTTA TAATTAA 3′ iv SA4-MTopF3.1 5′ GTATGATATTGTAAG (SEQ IDNO:6) GTATAATTTAATATTTTA TATATGT 3′ iv SA4-MTopF3.2 5′ AATATTGTATGATAT(SEQ ID NO:7) TGTAAGGTATAATTTAAT ATTTTA 3′ v SA4-MTopF45′ ATAAAATTATGGTTG (SEQ ID NO:8) AAATAATTGTATTATTTA TGA 3′ viiSA4-MTopF5 5′ AAACAAGTTGATTTA (SEQ ID NO:9) TATATTATGTATTAAATA ATGGAA 3′

The results of detecting the sequence-modified top strand of integratedSCCmec are shown in FIG. 1A (using SA4-MSRTop1 Scorpion™) and FIG. 1B(using SA4-MSRTop2 Scorpion™). The data indicate that the primers andprobes are specific for sequence-modified integrated SCCmec, and do notdetect unconverted integrated SCCmec within 45 cycles.

One Scorpion™ oligonucleotide (containing a forward primer and a probe)and a reverse primer were designed to detect the sequence-modified topstrand of the spa gene (Table 2). To determine the specificity of theprimers shown in Table 2 for the sequence-modified top strand of spagene from S. aureus, samples of “converted DNA” or “unconverted DNA”were analyzed in singleplex reactions. Using the same PCR conditionsdescribed above, except with primers/probe SA5-MSFTop1 (2 μM) (JOEdetection) and SA5-MTopR1 (2 μM), the results indicate that the primersand probes are specific for sequence-modified spa, and do not detectunconverted spa within 45 cycles (FIG. 2).

TABLE 2 Top Strand Assay Scorpions and Primers for spa Top StrandScorpion ™ or Primer Name (SEQ ID NO) Sequence SA5-MSFTop15′ quencher-ACCCCCAACAAATAT (SEQ ID NO:10) TACACCACCAAATATAACCCCCT-dye/blocker-TTACCTCTACCTATT CTATTTCTAATTTTACCTATA 3′ SA5-MTopR15′ CTTAAATCATCTTTAAAACTTTAA (SEQ ID NO:11) ATAAAACCA 3′

One Scorpion™ oligonucleotide (containing a forward primer and a probe)and a reverse primer were designed to detect the sequence-modified topstrand from the mecA gene (Table 3). To determine the specificity of theprimers shown in Table 3 for the sequence-modified top strand of mecAgene, samples of “converted DNA” or “unconverted DNA” were analyzed insingleplex reactions. Using the same PCR conditions described above,except with primers/probe SA1-MSFTop1 (2 μM) (FAM detection) andSA1-MTopR1 (2 μM), the results indicate that the primers and probes arespecific for sequence-modified mecA, and do not detect unconverted mecAwithin 45 cycles (FIG. 3).

TABLE 3 Top Strand Assay Scorpions and Primers for mecA Top StrandScorpion ™ or Primer Name (SEQ ID NO) Sequence SA1-MSFTop15′ quencher-AGCGCCATTATTTT (SEQ ID NO:12) CTAATACACTATAAATTAAAAAAATCTGGCGCT-dye/blocker-TTTAGG TTATGGATAAGGTGAAATATTGATT A 3′ SA1-MTopR1:5′ TCTTTATATATTTTATTTACAAC (SEQ ID NO:13) TTATTACATACCATCA 3′

Bottom Strand Assay

To detect the sequence-modified bottom strand of integrated SCCmec,seven forward primers and two Scorpion™ primer/probes containing areverse orfx primer and probe were designed. The forward primers weredesigned to detect the bottom strand of sequence modified integratedSCCmec types ii, iii, iv, v, and vii (Table 4). The forward primers wereeach specific to a single SCCmec type, but the Scorpion™ primer/probe,which hybridizes to orfx was not specific for a SCCmec type. Todetermine the specificity of the primers shown in Table 4 forsequence-modified bottom strand of integrated SCCmec, samples of“converted DNA” or “unconverted DNA” were analyzed in singleplexreactions using the PCR conditions described above, except that primersincluded: SA4-MFBot1 (2 μM), SA4-MFBot2 (2 μM), SA4-MFBot3 (2 μM),SA4-MFBot4 (2 μM), SA4-MFBot5.1 (2 μM (SA4-MSRBot1 or SA4-MSRBot2 (2 μM)(Cal Fluor Red 610 detection).

TABLE 4 Bottom Strand Assay Scorpions and Primers for Integrated SCCmecBottom Strand Scorpion ™ or SCCmec Primer Name MREJ Type (SEQ ID NO)Sequence Scorpion ™ SA4-MSRBot1 5′ quencher-AGCGCCA (types i. ii, (SEQID NO:14) TTTAATCCACCAATAACAA iii, iv. v, vii) ATACGGCGCT-dye/blocker-GAATTGAATTA ATGTATGATTTAAGGGTAA AGTGA 3′ Scorpion ™ SA4-MSRBot25′ quencher-AGCCGGC (types ii, iii, (SEQ ID NO:15) TACATTATAAAACATCCTTiv, v, vii) ATACAAACCGGCT dye/ blocker-GTGATTTTGTA TTTGTTATTGGTGGAT TA3′ i & ii SA4-MFBot1 5′ TTACTTAAAATAAAAA (SEQ ID NO:16)ACTACAAAAACTAACTATA TCAAA 3′ iii SA4-MFBot2 5′ CTCTATAAACATCATAT (SEQ IDNO:17) AATATTACAAAATATAATC CA 3′ iv SA4-MFBot3 5′ TAAAAACCACTACTAAA (SEQID NO:18) AAAAATATAAAAATCC A 3′ v SA4-MFBot4 5′ AACTCTACTTTATATT (SEQ IDNO:19) ATAAAATTACAACTAAAAT AACCA 3′ v SA4-MBotF4.1 5′ ACAACTAAAATAACCA(SEQ ID NO:20) CATCATTTATAATATACTT CT 3′ vii SA4-MFBot5.15′ ACTTACTACAAACATC (SEQ ID NO:21) TAATACAAAAAAAAATC AA 3′ viiSA4-MFBot5.2 5′ AAAAAAATCAATTTAC (SEQ ID NO:22) ACACCATATATTAAATAAT AA3′ vii SA4-MBot5.3 5′ CTCATATTTTTTAATT (SEQ ID NO:23) TTATTTATAATACACTTCT 3′ vii SA4-MBot5.4 5′ TTTTCTCATATTTTTT (SEQ ID NO:24)AATTTTATTTATAATACAC TTCT3′

The results of detecting the sequence-modified bottom strand ofintegrated SCCmec are shown in FIG. 4A (using SA4-MSRBot1 Scorpion™) andFIG. 4B (SA4-MSRBot2 Scorpion™). The data indicate that the primers andprobes arc specific for sequence-modified integrated SCCmec, and do notdetect unconverted integrated SCCmec within 45 cycles,

One Scorpion™ oligonucleotide (containing a forward primer and a probe)and a reverse primer were designed to detect the sequence-modifiedbottom strand of the spa gene from S. aureus (Table 5). To determine thespecificity of the primers shown in Table 5 for the sequence-modifiedbottom strand of spa gene from S. aureus, samples of “converted DNA” or“unconverted DNA” were analyzed in singleplex reactions. Using the samePCR conditions described above, except with primers/probe SA5-MSRBot1 (2μM) (JOE detection) and SA5-MBotF1 (2 μM), the results indicate that theprimers and probes are specific for sequence-modified spa, and do notdetect unconverted spa within 45 cycles (FIG. 5).

TABLE 5 Bottom Strand Assay Scorpions and Primers for spa Bottom StrandScorpion ™ or Primer Name (SEQ ID NO) Sequence SA5-MSRBot15′ quencher-AGCGGCAAATATTAAATATACCT (SEQ ID NO:25)AACTTAAACACTAATCGCCGCTdye/blocker-T TTGGATTATTTTTAAGGTTTTGGATAAAATTA 3′SA5-MBotF1 5′ ATCTAATAACATAACACCTACTACAATACTAC (SEQ ID NO:26) ACAA 3′

One Scorpion™ oligonucleotide (containing a forward primer and a probe)and a reverse primer were designed to detect the sequence-modifiedbottom strand from the mecA gene (Table 6). To determine the specificityof the primers shown in Table 6 for the sequence-modified top strand ofmecA gene, samples of “converted DNA” or “unconverted DNA” were analyzedin singleplex reactions. Using the same PCR conditions described above,except with primers/probe SA1-MSFBot1 (2 μM) (FAM detection) andSA1-MBotR1 (2 μM), the results indicate that the primers and probes arespecific for sequence-modified mecA, and do not detect unconverted mecAwithin 45 cycles (FIG. 6)

TABLE 6 Bottom Strand Assay Scorpions and Primers for mecA Bottom StrandScorpion ™ or Primer Name (SEQ ID NO) Sequence SA1-MSFBot15′ quencher-AGCGCCGTGTTTATAGATTGAAA (SEQ ID NO:27)GGATTTGTATTGGCGCT-dye/blocker-ACTGA TTCAAATTACAAACAAAATAAAATACTAA 3′SA1-MBotR1 5′ GTTTTTTAATAAGTGAGGTGTGTTAATATTGT (SEQ ID NO:28) TA 3′

EXAMPLE 3 Multiplex Amplification and Detection of Sequence-ModifiedNucleic Acids

A multiplex assay for sequence-modified spa, mecA, integrated SCCmec,and an internal control, was performed using the following Scorpion™oligonucleotides and primers. A 10× Primer Mix was prepared using 0.1×TEpH 8 as the diluent. Final concentrations of each component in the 10×mix indicated in parenthesis: SA5-MSFTop1 (2 μM); SA5-MTopR1 (2 μM);SA1-MSFTop1 (2 μM); SA1-MTopR1 (2 μM); SA4-MSRTop2 (2 μM); SA4-MTopF1.1(2 μM); SA4-MTop2.1 (2 μM); SA4-MTop3.1 (2 μM); SA4-MTopF4 (2 μM);SA4-MTopF5 (2 μM); IC-noloop-SFP-Dx2-DQ (1 μM); IC-SR4-Dx2 (1 μM). Thereaction mix for a single well of a 96-well optical PCR plate includedthe following: 12.5 μL 2× Master Mix; 2.5 μL of 10× Primer Mix(components listed above); 4.0 μL Nuclease-free water; 1.0 μL Internalcontrol amplicon; 5.0 μL bisulfite converted genomic DNA template (250pg). The conditions for the real time PCR run on a Applied BiosystemsAB7500 were as follows: Step 1: 95° C. 10 min; Step 2: 95° C. 15 sec;Step 3: 60° C. 35 sec. Steps 2-3 were repeated for a total of 45 cycles.Spa is detected with JOE, integrated SCCmec is detected with Cal FluorRed 610, mecA is detected with FAM, and the internal positive controlwas detected with Quasar 670.

The results are shown in FIG. 7 and indicate that the assay cansimultaneously positively identify sequence-modified nucleic acidscorresponding to target genes from sequence-modified MRSA containedwithin a clinical specimen, but not detect target genes from unmodifiedMRSA. The data shown here was produced by using a nasal swab clinicalspecimen known to be positive for MRSA. The nucleic acids in thespecimen were extracted using the QIAamp kit. Half of the genomicextraction was saved as “unmodified nucleic acids” and the other halfwas converted with HQS MethylEasy Fast™. Both sequence modified(converted) sample and unmodified sample were then used as template fora multiplex reaction using the primers and probes described above.

EXAMPLE 4 Detection of Unmodified spa and mecA

One Scorpion™ oligonucleotide (containing a forward primer and a probe)and a reverse primer were designed to detect the spa gene (Table 7).Using the same PCR conditions described above, except with primers/probeSA5-SF2A-DQS (2 μM) (Cal Fluor Red 610 detection) and SA5-R2c (2 μM).The results indicate that the primers and probes are capable ofdetecting spa in a MRSA sample (FIG. 8).

TABLE 7 Scorpions and Primers for Unmodified spa Top StrandScorpion ™ or Primer Name (SEQ ID NO) Sequence SA5-SF2A-DQS5′ quencher-AGGCCACCAGATATAAGTAATGT (SEQ ID NO:29)ACCTAAAGTGGCCT-dye/blocker-ATTCGTAA ACTAGGTGTAGGTATTGCA 3′ SA5-R2C5′ ACTTGATAAAAAGCATTTTGTTGAGCT 3′ (SEQ ID NO:30)

One Scorpion™ oligonucleotide (containing a forward primer and a probe)and a reverse primer were designed to detect the mecA gene (Table 8).Using the same PCR conditions described above, except with primers/probeSA1-SF11C-DQS (2 μM) (FAM detection) and SA1-R11.2 (2 μM). The resultsindicate that the primers and probes are capable of detecting mecA in aMRSA sample (FIG. 9).

TABLE 8 Scorpions and Primers for Unmodified mecA Top Strand ScorpionTMor Primer Name (SEQ ID NO) Sequence SA1-SF11C-DQS5′ quencher-AGCCGCTATAGATTGAAAGGATC (SEQ ID NO:31)TGTACTGGCGGCT-dye/blocker-AGGTTACGG ACAAGGTGAAATACTGA 3′ SA1R11.25′ GTGAGGTGCGTTAATATTGCCATTA 3′ (SEQ ID NO: 32)

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other embodiments are set forth within the following claims.

1. A method for detecting Staphylococcus aureus in a biological samplecomprising: (a) converting the non-methylated cytosines present in thenucleic acids contained in a biological sample, to uracils to producesequence-modified nucleic acids, (b) bringing the biological samplecontaining the sequence modified nucleic acids in contact with one ormore primer pairs selected from the group consisting of: (i) a firstprimer pair which is complementary to a segment of a marker genespecific for Staphylococcus aureus of the sequence modified nucleicacids; (ii) a second primer pair which is complementary to a segment ofthe mecA gene of the sequence modified nucleic acids; and (iii) a thirdprimer pair, one primer of which is complementary to a segment of SCCmecof the sequence modified nucleic acids and the other primer of which iscomplementary to a segment of the orfx gene of the sequence modifiednucleic acids; under conditions wherein the primers specificallyhybridize and amplification products of the sequence-modified nucleicacids are produced; and (c) identifying the modified nucleic acids fromStaphylococcus aureus by detecting the amplification product produced byone or more of the primer pairs.
 2. The method of claim 1, wherein thebiological sample is contacted with the first primer pair, the secondprimer pair, and the third primer pair in a multiplex amplificationreaction.
 3. The method of claim 1, wherein the marker gene specific forStaphylococcus aureus is selected from the group consisting of: spa,agr, ssp protease, sir, sodM, cap, coa, alpha hemolysin, gammahemolysin, femA, Tuf, sortase, fibrinogen binding protein, clfB, srC,sdrD, sdrE, sdrF, sdrG, sdrH, NAD synthetase, sar, sbi, rpoB, gyrase A,and orfX.
 4. The method of claim 3, wherein the marker gene specific forStaphylococcus aureus is spa.
 5. The method according to claim 1,wherein the converting step is accomplished by contacting the nucleicacids with sodium bisulfite.
 6. The method according to claim 1, whereinone or more of the primers is degenerate.
 7. The method according toclaim 1, wherein said detecting is accomplished using a labeledoligonucleotide probe for each amplification product.
 8. The methodaccording to claim 7, wherein the amplification is performed using realtime PCR.
 9. The method according to claim 8, wherein, for eachamplification product to be detected, the probe and one of the primersof the primer pair are part of the same molecule.
 10. The methodaccording to claim 1, wherein one or both of the primers of the firstprimer pair comprises a sequence selected from the group consisting ofSEQ ID NO:10-11.
 11. The method according to claim 1, wherein one orboth of the primers of the second primer pair comprises a sequenceselected from the group consisting of SEQ ID NO:12-13.
 12. The methodaccording to claim 1, wherein one or both of the primers of the thirdprimer pair comprises a sequence selected from the group consisting ofSEQ ID NO:1-9.
 13. The method according to claim 1, wherein one or bothof the primers of the first primer pair comprises a sequence selectedfrom the group consisting of SEQ ID NO:25-26.
 14. The method accordingto claim 1, wherein one or both of the primers of the second primer paircomprises a sequence selected from the group consisting of SEQ IDNO:27-28.
 15. The method according to claim 1, wherein one or both ofthe primers of the third primer pair comprises a sequence selected fromthe group consisting of SEQ ID NO: 14-24.
 16. A method for determiningif a biological sample from an individual contains methicillin resistantStaphylococcus aureus (MRSA) or methicillin sensitive Staphylococcusaureus (MSSA), comprising: (a) converting the non-methylated cytosinespresent in the nucleic acids contained in the biological sample, touracils to produce sequence-modified nucleic acids, (b) bringing thebiological sample containing the sequence modified nucleic acids incontact with: (i) a first primer pair which is complementary to asegment of a marker gene specific for Staphylococcus aureus of thesequence modified nucleic acids; (ii) a second primer pair which iscomplementary to a segment of the mecA gene of the sequence modifiednucleic acids; and (iii) a third primer pair, one primer of which iscomplementary to a segment of SCCmec of the sequence modified nucleicacids and the other primer of which is complementary to a segment of theorfx gene of the sequence modified nucleic acids; under conditionswherein the primers specifically hybridize and amplification products ofthe sequence-modified nucleic acids are produced; and (c) identifyingthe modified nucleic acids from Staphylococcus aureus by detecting theamplification product produced by one or more of the primer pairs,wherein i) amplification of all three sequence-modified nucleic acidsindicates MRSA in the sample; and ii) amplification of the S. aureusspecific marker gene alone, or integrated SCCmec and the S. aureusspecific marker gene, but not mecA, indicates MSSA in the sample. 17.The method of claim 16, wherein the marker gene specific forStaphylococcus aureus is selected from the group consisting of: spa,agr, ssp protease, sir, sodM, cap, coa, alpha hemolysin, gammahemolysin, femA, Tuf, sortase, fibrinogen binding protein, clfB, srC,sdrD, sdrE, sdrF, sdrG, sdrH, NAD synthetase, sar, sbi, rpoB, gyrase A,and or orfX.
 18. The method according to claim 16, wherein theconverting step is accomplished by contacting the nucleic acids withsodium bisulfite.
 19. The method according to claim 16, wherein one ormore of the primers is degenerate.
 20. The method according to claim 16,wherein said detecting is accomplished using a labeled oligonucleotideprobe for each amplification product.
 21. The method according to claim20, wherein, for each amplification product to be detected, the probeand one of the primers of the primer pair are part of the same molecule.22. The method according to claim 16, wherein one or both of the primersof the first primer pair comprises a sequence selected from the groupconsisting of SEQ ID NO:10-11.
 23. The method according to claim 16,wherein one or both of the primers of the second primer pair comprises asequence selected from the group consisting of SEQ ID NO:12-13.
 24. Themethod according to claim 16, wherein one or both of the primers of thethird primer pair comprises a sequence selected from the groupconsisting of SEQ ID NO:1-9.
 25. The method according to claim 16,wherein one or both of the primers of the first primer pair comprises asequence selected from the group consisting of SEQ ID NO:25-26.
 26. Themethod according to claim 16, wherein one or both of the primers of thesecond primer pair comprises a sequence selected from the groupconsisting of SEQ ID NO:27-28.
 27. The method according to claim 16,wherein one or both of the primers of the third primer pair comprises asequence selected from the group consisting of SEQ ID NO:14-24.
 28. Amethod of identifying a methicillin-resistant Staphylococcus aureus(MRSA) or methicillin sensitive Staphylococcus aureus (MSSA), ifpresent, in a biological sample, comprising: (a) bringing the biologicalsample in contact with: (i) a first primer pair which is complementaryto a marker gene specific for Staphylococcus aureus; (ii) a secondprimer pair which is complementary to the mecA gene; and (iii) a thirdprimer pair, one primer of which is complementary to SCCmec and theother primer of which is complementary to the orfx gene; underconditions wherein the primers specifically hybridize and amplify themarker gene, mecA gene, SCCmec and orfx gene, and (b) identifying theMSSA and/or MRSA by detecting an amplification product produced by allof the three primer pairs, wherein i) amplification of all threesequence-modified nucleic acids indicates MRSA in the sample; and ii)amplification of the S. aureus specific marker gene alone, or integratedSCCmec and the S. aureus specific marker gene, but not mecA, indicatesMSSA in the sample.
 29. The method of claim 28, wherein the marker genespecific for Staphylococcus aureus is selected from the group consistingof: spa, agr, ssp protease, sir, sodM, cap, coa, alpha hemolysin, gammahemolysin, femA, Tuf, sortase, fibrinogen binding protein, clfB, srC,sdrD, sdrE, sdrF, sdrG, sdrH, NAD synthetase, sar, sbi, rpoB, gyrase A,and orfX.